Structure analysis and expressions of a novel tetratransmembrane protein,lysosoma-associated protein transmembrane 4β associated with hepatocellular carcinoma

AIM: To analyze the structure and expressions of the protein encoded by an HCC-associated novel gene, lysosomeassociated protein transmembrane 4 β (LAPTM4B). METHODS: Primary structure and fundamental characteristics of LAPTM4B protein were analysed with bioinformatics. Expressions of LAPTM4B in HCC tissues and various cell lines were detected using polyclonal antibodies and Western blot. RESULTS: LAPTM4B encoded two isoforms of proteins with molecular masses 35-ku and 24-ku, respectively. The expression level of LAPTM4B-35 protein in HCC tissues was dramatically upregulated and related to the differentiation status of HCC tissues, and it was also high in some cancer cell lines. Computer analysis showed LAPTM4B was an integral membrane protein with four transmembrane domains. LAPTM4B showed relatively high homology to LAPTM4A and LAPTM5 in various species. CONCLUSION: LAPTM4B gene encoded two isoforms of tetratansmembrane proteins, LAPTM4B-35 and LAPTM4B-24. The expression of LAPTM4B-35 protein is upregulated and associated with poor differentiation in human HCC tissues, and also at high levels in some cancer cell lines. LAPTM4B is an original and conserved protein.

Expression of lysosome-associated protein transmembrane 4B-35 in cancer and its correlation with the differentiation status of hepatocellular carcinoma

AIM: To produce high-quality polydonal antibody to lysosome associated protein transmembrane 4B-35 and to identify LAPTM4B-35 expression in cancer tissues and its correlation with differentiation status of hepatocellular carcinoma (HCC).METHODS: The 297 bp 5' end of LAPTM4BcDNA was obtained by PCR and inserted into prokaryotic expression vector pGEXKG. Then the recombinant pGEX-KG-N1-99 was transformedinto E. coli JM109 to express GST-fusion protein. The fusion protein was purified by glutathione sepharoseTM 4B agarose. The purified GST-LAPTM4B-N1-99 was characterized by SDSPAGE, and used to immunize rabbits. The titer and specificity of antisera were detected by ELISA and Western blot, respectively. The correlation between the expression levels of LAPTM4B-35 and the differentiation status of HCC was analyzed via Western blot. The expression of LAPTM4B35 in HCC and other six cancer tissues was investigated via tissue chip and immunohistochemical analysis. RESULTS: About 6.2 mg of pure GST-LAPTM4B-N1-99 was isolated from 1 L of bacteria. The GST-LAPTM4B-N1-99produced high titer antisera in rabbits and showed good immunity. Western blot showed specific reactions for the antibody to the LAPTM4B-35 in the total proteins from HCC tissues and BEL-7402 cells, also to the fusion protein purified or in the transformed bacteria. LAPTM4B-35 was remarkably expressed in several cancers, such as HCC, breast cancer, gastric carcinoma, lung cancer, and colon carcinoma, but not commonly expressed in esophageal cancer and rectum carcinoma. Notably, the expression levels of LAPTM4B-35 were significantly and inversely correlated to the differentiation of HCCs in a 20 case analysis. CONCLUSION: Specific polyclonal antibody (LAPTM4B-N1-99-pAb) to LAPTM4B-35 was produced. It identified the expression of LAPTM4B-35 in some cancer tissues originated from single layer cuboidal and columnar epithelial cells and firmly demonstrated that the expression of LAPTM4B-35 in HCC was inversely correlated with the differentiation of HCC.